The dynamics and stabilities (i.e., physicochemical properties) of proteins and protein complexes are dictated by the solution environment. Changes in the solution environment may alter the distribution of states present in solution (i.e., the free-energy landscape) and thus their physicochemical properties. This work examines the effects of temperature (4 °C, 21 °C, and 37 °C), electrospray ionization buffers (ammonium acetate (AmAc) and triethylammonium acetate (TEAA)), and solvent (H2O and D2O) on the physicochemical characteristics of the myoglobin/heme complex using limited proteolysis (i.e., trypsin digestion) and ion mobility-mass spectrometry (IM-MS). Electrospray ionization mass spectra of peptides formed by limited proteolysis showed increased peptide abundances at higher temperatures and in the absence of buffer molecules relative to the intact precursors. Interestingly, peptide abundances in D2O are more consistent across the temperature range studied relative to the peptide abundances in H2O, which demonstrates that D2O not only stabilizes proteins but acts as a kinetic trap for specific protein conformations. As an example, buffer components and D2O stabilize the myoglobin/heme interaction, as is evidenced by the modulation of peptide 64–77, an α-helical region that interacts with heme, and by shifts in the abundances of unfolded protein precursors. Lastly, IM-MS analysis of digested peptides suggests that AmAc alters the tertiary structure of the myoglobin/heme complex, whereas TEAA alters the secondary structure of the complex. The preservation of secondary structure in TEAA is further illustrated by the shift in arrival time distributions of 1+ ions in TEAA relative to the 2+, 3+, 4+, and 5+ ions present in other buffer conditions. Overall, the data show that buffer-solvent interactions govern the hydration of myoglobin and consequently, the physicochemical properties of the complex.
Carter Lantz, Leila Minian, Roza Avetisyan et al.· ACS Measurement Science Au· 0 citations
Buffers are commonly selected for their compatibility with biochemical measurements or their specific capabilities, yet buffer interactions with proteins and the surrounding water can actively reshape protein activity, structure, and dynamics. Here, we compare the influences of three widely used electrospray ionization (ESI) buffers on the dynamics and stability of wild-type transthyretin (wtTTR) and TTR mutants (V30M, L55P, T119M, V122I) using native mass spectrometry (nMS). Ammonium acetate (AmAc), ethylenediammonium diacetate (EDDA), and triethylammonium acetate (TEAA) are commonly employed in nMS because they stabilize the sample in solution, facilitate gentle ionization, and minimize adduct formation on proteins and protein complexes. In comparison to AmAc, EDDA and TEAA reduce the average charge state (Zavg) of ions, consistent with conformational changes that decrease the solvent-accessible surface area (SASA) of proteins. Intact protein hydrogen–deuterium exchange experiments indicate significantly lower deuterium uptake for all TTR proteoforms in EDDA compared with AmAc or TEAA. Ion mobility MS revealed that each TTR proteoform has a larger average CCS and broader CCS distributions in AmAc, indicating greater conformational heterogeneity and dynamics than in EDDA or TEAA. Measurements of TTR tetramer disassembly and reassembly further demonstrated that the buffer identity strongly influences tetramer stability in solution. The buffer-dependent effects arise from differences in buffer-protein and buffer-solvent interactions that alter the hydration of the protein. These results highlight that buffer composition can significantly influence experimentally observed protein dynamics and stability, with important implications for interpreting measurements across biochemical and biophysical techniques.
Emily Burningham, Carter Lantz, R. Rider et al.· Analytical Chemistry· 0 citations
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